A conventional functional assay system for assessing cellular delivery of an antisense agent is usually based on down-regulation of the translational step in gene expression by the antisense agent. The procedure suffers from several theoretical as well as practical problems. In the proposed study, the PI plans to devise a cellular assay system in which an antisense agent up-regulates the protein synthesis in such a way that a new protein, luciferase in the present case, begins to appear as the antisense agent enters the cell. The system can be developed by transfecting cells with a luciferase plasmid with aberrant splicing that can be corrected by an antisense agent. At least three different recombinant luciferase plasmids will be prepared by inserting a mutant human b-globin intron 2 at different locations of a luciferase plasmid pTRE-LUC from a commercial source. Recently, the PI has shown that the aberrant splicing caused by the mutant intron can be corrected by a 17-mer antisense targeted to its 5' splice site. In a preliminary experiment, the PI found that the HeLa cells transfected by the recombinant luciferase plasmid indeed produce luciferase when the oligomer was introduced to the cell by Lipofectamine, clearly establishing feasibility. In addition to HeLa cells, fibroblast cell line NIH/3T3 and monocyte-derived Raw 264.7 cells will be transfected with the plasmid. These cells will demonstrate different endocytic activities. When developed, the present system will be most useful and could become a universal assay system in objectively comparing various novel strategies of antisense/antigen delivery. Due to its intrinsic nature, however, ther system cannot be used with an oligomer that activates RNase H. However, this is not a limitation.